Evaluating the Effects of Mixed Osteolytic/Osteoblastic Metastasis on Vertebral Bone Quality in a New Rat Model Lisa Wise-Milestone, 1 Margarete K. Akens, 1 Thomas J. Rosol, 2 Seyed-Parsa Hojjat, 1 Marc D. Grynpas, 3 Cari M. Whyne 1 1 Orthopaedic Biomechanics Laboratory, Sunnybrook Research Institute, UB-19, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3M5, 2 Department of Veterinary Biosciences, The Ohio State University, 307 Goss Lab, 1925 Coffey Road, Columbus, Ohio, 3 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5 Received 23 June 2011; accepted 6 October 2011 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.21577 ABSTRACT: Spinal metastases often show mixed areas of enhanced (osteoblastic) bone growth adjacent to areas of thinning (osteolytic) bone. This study aims to quantitatively characterize bone quality and tumor burden within a new rat model of mixed osteolytic/ osteoblastic spinal metastases. Mixed vertebral metastases were analyzed in nude rats 21-days post intracardiac injection of Ace-1 canine prostate cancer cells. Vertebral micro-architecture was assessed in mCT images. Histologic processing quantified tumor burden (PTHrP), osteoclast activity (TRAP), and osteoid formation (Goldner’s Trichrome) in ½ of all samples. Remaining samples were mechan- ically tested to failure in compression. Metastatically involved vertebrae exhibited extreme osteolysis, evident through an increase in osteoclasts leading to significantly reduced trabecular bone volume. Metastatically involved vertebrae also exhibited increased osteoid characteristic of osteoblastic lesions. While mechanical properties in tumor-bearing vertebrae were not significantly decreased compared to controls, a strong correlation was found between trabecular volumetric BMD and ultimate force. The highly aggressive Ace-1 skeletal metastases demonstrated predominant osteolysis with some areas of immature, new osteoblastic bone formation. Bone quality resulting from these lesions consisted of decreased structural properties, but without a significant impact on mechanical integrity. ß 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res Keywords: skeletal metastasis; osteolytic/osteoblastic; vertebral bone quality; rat model Metastases develop in the spine of up to one-third of all cancer patients, compromising the mechanical in- tegrity of the vertebrae and increasing the risk of pathological fractures. 1 Local interactions between bone and tumor cells form a vicious cycle 2 that can lead to osteolytic bone destruction, osteoblastic excess bone deposition, or mixed osteolytic/osteoblastic lesions. 3 Tumor-derived factors stimulate osteoblastic bone for- mation, causing hyperactive osteoblasts to form wide trabeculae of immature woven bone with a poorly organized microstructure and an elevated risk of pathological fracture. 3 Persistent bone resorption from osteolytic lesions results in a higher rate of pathologic fracture even in the absence of a traumatic event. 3,4 Widespread use of systemic and local therapies aimed at skeletal lesions has revealed a changing pattern of metastatic disease from classic osteolytic lesions to considerably more mixed osteolytic/osteoblastic disease with more diffuse involvement. 5 It has been suggested that metastases may dynamically evolve between osteoblastic and osteolytic patterns with diagnostic tools capturing only a static representation of the prevalent pattern. 2 The net response of bone exposed to both osteolytic and osteoblastic factors is unpre- dictable, yet the effects of mixed lesions on bone quali- ty have not been adequately studied to date. Thorough insight into the underlying bone quality of mixed skeletal metastases is necessary in order to quantify fracture risk and to effectively assess the effects of new and existing therapeutics and direct their use in bones with mixed lesions. Many animal models of pure osteolytic 6,7 or osteoblastic 8,9 disease have been well established to study skeletal metasta- ses and both local and systemic therapies for these pa- thologies. Animal models that are representative of mixed disease have been less commonly studied. The PA-III cell line, derived from a spontaneous prostate adenocarcinoma in a Lobund Wistar rat, has been shown to induce both osteolytic and osteoblastic reac- tions when transplanted adjacent to bone. 10 However, this means of tumor cell administration does not clini- cally represent a true metastasis scenario. The MAT- Ly-Lu subline of the rat Dunning prostate carcinoma (R33227) model presents mixed lesions, but despite having an osteoblastic origin, these bone metastases are unpredictable and often present mainly osteolytic features. 8,9 Other reported models of mixed osteolytic/ osteoblastic skeletal metastases include the MHMX unclassified human sarcoma, 11 TSU-PR1, 12 and C4-2B 13 cell lines, but have had limited success in the rat. Recently, a nude mouse model with mixed bone me- tastases has been established using a canine prostate carcinoma xenograft (Ace-1). 14 Following intra-tibial injections, 70% of mice developed areas of osteolysis and new woven bone on the endosteal and periosteal surfaces of tibiae, fibulae, and femora. Intracardiac in- jection of Ace-1 cells resulted in mixed bone metasta- ses in all injected mice, most commonly in the proximal humeri, distal femora, proximal tibiae, ribs, and lumbar vertebrae. One of the most significant limitations of the mouse model is the relatively small size of its skeleton, pre- senting challenges with certain analytical techniques. In particular, destructive testing in order to determine Additional Supporting Information may be found in the online version of this article. Correspondence to: Cari M. Whyne (T: 416-480-5056; F: 416-480- 5856; E-mail: cari.whyne@sunnybrook.ca) ß 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. JOURNAL OF ORTHOPAEDIC RESEARCH 2011 1